WO1987007147A1 - Methode pour produire des anticorps monoclonaux a un complexe immunogenique anticorps-antigene - Google Patents

Methode pour produire des anticorps monoclonaux a un complexe immunogenique anticorps-antigene Download PDF

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Publication number
WO1987007147A1
WO1987007147A1 PCT/US1987/001295 US8701295W WO8707147A1 WO 1987007147 A1 WO1987007147 A1 WO 1987007147A1 US 8701295 W US8701295 W US 8701295W WO 8707147 A1 WO8707147 A1 WO 8707147A1
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WO
WIPO (PCT)
Prior art keywords
monoclonal antibody
antigen
fragment
immunogenic complex
complex
Prior art date
Application number
PCT/US1987/001295
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English (en)
Inventor
William Jackson Payne, Jr.
Original Assignee
Murex Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
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Publication of WO1987007147A1 publication Critical patent/WO1987007147A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins

Definitions

  • the present invention relates to antibodies and methods for their production. More particularly, the present invention comprises a method for the production of monoclonal antibodies designed to recognize the juncture between an immunogenic antibody:antigen complex.
  • an assay method in which a first antibody recognizes and binds to an analyte, typically an antigen, suspected of being in a solution.
  • a labeled second antibody a monoclonal, recognizes and binds preferentially to the juncture between the first antibody, and any of the analyte present, yet will not bind to analyte alone or antibody alone.
  • the presence or amount of labeled material present is measured and the quantity of analyte present determined.
  • the assay is unique in that it is highly selective: only material bound to the first antibody will be recognized, and only the juncture will be bound by the second antibody. This selectivity eliminates a substantial amount of false or spurious signal resulting from the cross-reactive binding of labeled antibody to materials other than the analyte or to analyte not bound by the first antibody.
  • the present invention relates to a method for the production of monoclonal antibodies to a unique antigen. More particularly, disclosed is a method for producing a monoclonal antibody directed against the juncture of the idiotypic determinant of a first monoclonal antibody and the antigenie determinant of an antigen when the monoclonal antibody and the antigen are bound together, comprising: (a) providing an antigen of interest; (b) subjecting the antigen to limited proteolytic digestion to produce one antigenic fragment capable of binding to an antibody directed against the antigen; (c) providing a first monoclonal antibody directed against the antigen and/or antigenic fragment; (d) separating the Fab or
  • step (f) injecting the immunogenic complex of step (e) into a host animal; and (g) obtaining the monoclonal antibodies directed against the immunogenic complex capable of recognizing the juncture between the antigen and the first antibody when complexed together.
  • the method of the present invention is useful for the production of highly selective monoclonal antibodies to a unique antigenic entity.
  • an antigen of interest is subjected to limited proteolysis so as to provide, among other components, a fragment that contains an active antigenic determinant.
  • a monoclonal antibody directed against the antigenic determinant is cleaved enzymatically and Fab or F(ab') and Fc fragments are separated.
  • the antigen fragment and the Fab or F(ab') fragment are then combined so as to produce a complex possessing immunogenic properties.
  • Animal recipients are immunized with this immun ⁇ gen.
  • Specific antibody-producing lymphocytes are fused with myeloma cells and monoclonal antibodies are selected.
  • the resulting monoclonal antibody will selectively bind to the juncture of the antigen and the antibody, but not the individual components alone. Analyte selection will dictate the extent of proteolytic digestion.
  • a small antigen such as a hormone or drug, has one antigenic determinant, i.e., a sequence of amino acids or a molecular structure which is recognized by an antibody.
  • the desired epitope structure on a small antigen or antigen fragment has a higher probability of being recognized than if present on a large antigen with multiple antigenic determinants. Therefore, a large antigen may be degraded into a smaller fragment, which under optimum circumstances possesses only the antigenic determinant desired.
  • SOS polyacrylamide gel electrophoresis
  • PAGE polyacrylamide gel electrophoresis
  • Protein fragments are transferred to nitrocellulose paper by the Western blot technique, which is well known in the art. Blots are developed using a specific monoclonal antibody in an indirect enzyme-linked im unosorbent assay (“ELISA”) to identify the polypeptide fragment that contains the antigenic determinant. The identified fragment with the antigenic determinant is purified by molecular exclusion chromatography, or alternatively, 5 by SDS-PAGE followed by electroelutio ⁇ .
  • ELISA enzyme-linked im unosorbent assay
  • a monoclonal antibody is used that is capable of binding to the antigenic fragment.
  • the Fc fragment is removed from the antibody
  • the antibody can be cleaved just above the hinge region by using the enzyme papain, yielding a larger Fc fragment and two Fab fragments, each with a molecular weight of 50,000 daltons and each having a single antigen binding site.
  • the Fab fragment will be discussed in this application.
  • the F(ab') or Fab fragment can be purified by using either molecular exclusion or affinity (Protein A) chromatography; both methods are discussed in detail in the Examples set forth below.
  • an 5 antibody:antigen complex In order to create the im unogen necessary for immunization an 5 antibody:antigen complex must first be formed.
  • the purified antigen preparation possessing the desired antigenic determinant is combined with the Fab fragment of the antibody under appropriate conditions, i.e., pH, molarity and temperature.
  • the antigenic determinant is recognized and bound by the binding site of the Fab creating a more
  • the complex is purified by a means gentle enough to allow the complex to remain intact; for example, one appropriate method is molecular l ⁇ exclusion chromatography, which will separate the complex from the reactants based upon molecular size.
  • Immunogenicity the characteristic of a molecule or substance to evoke a hpst immune response can be enhanced by various means.
  • Existing methods include chemical cross-linking (glutaraldehyde) and
  • any antibodies used to create the conjunction site in the method should be produced from genetically identical animals as those used to make the conjunction binding antibodies. This reduces the possibility that a recipient will process an antibody as a foreign antigen, such as a Balb/c mouse recognizing a C57BL mouse antibody as foreign and producing antibodies to the antibody, rather than to the antigen-antibody conjunction as desired.
  • Monoclonal antibodies are prepared essentially as described by Milstein and Kohler (Nature, 256:495-497, 1975). The process is well known and is reviewed in the Examples set forth below. Briefly, an appropriate animal, such as a mouse, is injected with an immunogen, such as the complex of antigen and antibody fragments. Subsequently, the mouse is sacrificed and cells removed from its spleen are fused with myeloma cells. Fusion results in the formation of immortal hybrid cells, known as "hybridomas," which synthesize antibodies. The hybridoma population produced is screened to identify the desired antibodies.
  • the hybridoma populations thus identified are cloned by limiting dilution to isolate hybridomas derived from a single cell and thereafter are designated as monoclonal hybridoma.
  • the antibody obtained via this procedure is the product of a single B-cell from the immunized recipient animal that is generated in response to a specific antigenic " determinant identified on the immunogen. In the case described it is the juncture which is recognized.
  • the conjunction immunogen is injected into a suitable animal, such as a mouse.
  • the resulting hybridoma cells are screened, such as by ' Microtiter ELISA technique, for the production of a monoclonal antibody that binds to an intact (non-digested) antigen-antibody complex, but not the- antibody or the intact antigen alone.
  • the monoclonal antibody thus produced can be utilized in an assay procedure to bind to any co plexed antigen:antibody present in the reaction mixture.
  • Either this monoclonal antibody or the complexed monoclonal antibody can be tagged with one of manypossible labeling materials, such as an enzyme, fluorescent, chemiluminescent, bioluminescent, isotopic, ferromagnetic, or other common signal generating material.
  • labeling materials such as an enzyme, fluorescent, chemiluminescent, bioluminescent, isotopic, ferromagnetic, or other common signal generating material.
  • An alternative embodiment involves first forming a complex of intact antigen and intact first monoclonal antibody. Then the Fc fragment is cleaved from the antibody using pepsin or papain, followed by limited protolytic digestion of the antigen under conditions that will not result in digestion of the bound antibody. The purified product is then used as the new immunogen to be injected into the recipient animal. Care must be taken to create reaction conditions conducive to the desired reaction. If the pH is too low dissociation of the antigen:antibody complex is possible. Furthermore, because papain and pepsin are relatively non-specific enzymes (other than the action on sulfhydryl groups) there is the possibility of degradation of the antigen.
  • Chlamydia trachomi is major outer membrane protein (MOMP) as the antigen:
  • MOMP major outer membrane protein
  • Procedure for limited proteolytic digestion of antigen Purified (MOMP) eluted electrophoretically from gels is dissolved at approximately 0.5 mg/ml in sample buffer which contains 0.125 M Tris/HCl at pH 6.8, 0.5% SDS,
  • proteases include, but are not limited to, endoproteinase Glu-C from Staphylococcus aureus V8, endoproteinase Arg-C from mouse submaxillary gland, endoproteinase Tys-C from Lysobacter enzymogenes, and the like.
  • proteolysis is stopped by boiling the samples for 2 minutes. 20 to 30ul (10 to 15ug) of each sample is loaded into a sample well of 15% acrylamide gel and the gel is run according to accepted procedures.
  • SDS-polyacrylamide gel electrophoresis of the digest is performed.
  • the preparations are transferred electrophoretically to nitrocellulose paper using the method of Towbin, et al , commonly referred .to as Western blot.
  • Towbin, et al commonly referred .to as Western blot.
  • To identify the polypeptide fragment of interest an ELISA is performed using a selected monoclonal antibody.
  • MOMP fragment i puri fied by SDS-PAGE and el ectroel uti on .
  • Purified antigen fragment and Fab preparations are mixed in a 20 mM phosphate buffer pH 7.4, with 1,50 mM NaCl. The mixture is allowed to incubate for a period of time until equilibrium is reached for the binding of the components. The incubation time- is determined by empirical observation. When equilibrium has been reached the mixture is subjected to molecular exclusion chromatography using, for example, Sephadex G-75 superfine gel filtration beads (Sigma, St. Louis, MO). Since the molecular weights of both the antigen fragments and the Fab fragments are known, fractions from a calibrated molecular exclusion column containing the complex are easily located by determining protein content using a spectrophotometric reading at 280 nm. The appropriate fractions are pooled and may be concentrated for use as an immunogen.
  • the monoclonal antibodies are prepared by fusing spleen cells, from a mouse immunized against- the chlamydia antigen:antibody complex, with an appropriate murine myeloma cell line (P3X63-Ag8.653). The resultant product is then cultured in a standard hypoxanthi ⁇ e, aminopterin, and thymidine (HAT) medium. Immunoassays are utilized to screen the specific antibodies produced by the growing hybridoma cultures. Typical immunoassays that may be used include microtiter ELISA and dot-blot ELISA. The first is done in 96 well microtiter plates and the second is done on strips of nitrocellulose paper.
  • the antibody alone, the antigen alone and the antibod :antigen complex are applied to the plates or paper as the antigen source for the conjunction binding antibody.
  • a second antibody i.e., enzyme conjugated anti-mouse IgG, is added to indicate where the desired first antibody-.antigen complex and not the antibody or antigen alone will be selected.
  • the immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present invention will be described in connection with mice.
  • the mouse is immunized by several injections of the immunogenic complex generally over a period of several weeks. When the mouse shows sufficient antibody titre against the immunogenic complex, as determined by conventional assay methods, it is given a booster injection of the immunogenic complex, then sacrificed- and the spleen is removed. The fusion is then initiated utilizing spleen cells from immunized mice and an appropriate myeloma cell line (For Example, P3X63-Ag8.653).
  • the hybridomas yielding antibodies specific for the immunogenic complex are selected and cloned utilizing standard methods.
  • the monoclonal antibodies from the clones are then tested to determine their specificity for the particular immunogenic complex.
  • Selected monoclonal antibodies specific for the immunogenic complex are then conjugated with an appropriate label.
  • Amounts of antibody sufficient for labeling and subsequent commercial production are obtained from monoclonal hybridomas by the accepted techniques, such as a batch or continuous tissue culture or culture in vivo in mammals, such as mice.
  • the monoclonal antibodies may be labeled with signal generating material, such as an enzyme, fluorescent compound, luminescent compound, radioactive compound, ferromagnetic label, and the like.
  • enzymes utilized as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase, urease, and the like. Conjugation with enzymes can be accomplished by any one of the conventional and accepted methods, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, the periodate method, or a biotin-a ' vidin bridge.
  • Example 2 The procedures according to Example 1 are followed except that in Step B the F(ab') antibody fragment is prepared rather than the Fab fragment using the following procedure: F(ab') pivotpreparation and purification:
  • Examples 4-7 The procedure according to Examples 1 or 2 is followed wherein substituted for Chlamydia trachomatis is one of the following antigens and substituted for the first monoclonal antibody directed against Chlamydia trachomatis is a monoclonal antibody directed against the selected antigen: Candida albicans, Human T-Lymphotropic Virus - III, Giardia Iambiia, or glycosylated hemoglobin.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
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Abstract

Méthode pour la préparation d'un anticorps monoclonal capable de reconnaître la jonction d'un déterminant antigénique et d'un site de liaison d'anticorps correspondant lorsqu'ils sont combinés l'un à l'autre. L'antigène peut être soumis à une digestion protéolytique limitée en vue d'obtenir un fragment immunochimiquement actif. De la même manière, un anticorps monoclonal dirigé contre le déterminant antigénique à étudier est enzymatiquement clivé pour obtenir le fragment Fab ou F(ab')2. Les deux fragments sont combinés de manière à former un complexe. Ce complexe immunogénique est ensuite injecté dans un animal récepteur et les anticorps produits reconnaissent spécifiquement la jonction du complexe immunogénique.
PCT/US1987/001295 1986-05-28 1987-05-28 Methode pour produire des anticorps monoclonaux a un complexe immunogenique anticorps-antigene WO1987007147A1 (fr)

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US86829786A 1986-05-28 1986-05-28
US868,297 1986-05-28

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0264219A2 (fr) * 1986-10-09 1988-04-20 Syntex (U.S.A.) Inc. Récepteurs pour des complexes immunologiques, leurs procédés de préparation et méthodes d'essais les utilisant
EP2168985A1 (fr) 2008-09-30 2010-03-31 Siemens Healthcare Diagnostics Products GmbH Anticorps destinés à la détermination du fragment de prothrombine F2/F1+2 dans un test d'immunité homogène
US20110086364A1 (en) * 2008-06-12 2011-04-14 Teknologian Tutkimuskeskus Vtt Detection of cannabis use

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4427782A (en) * 1981-03-03 1984-01-24 Caldwell Harlan D Isolation of principal outer membrane protein and antigen of Chlamydia trachomatis
US4544640A (en) * 1982-04-09 1985-10-01 Fujizoki Pharmaceutical Co., Ltd. Anti immune complex antibody for determining SLE, rheumatoid arthritis or tetanus
US4636478A (en) * 1984-07-16 1987-01-13 Becton, Dickinson And Company Monoclonal antibodies recognizing L-thyroxine
US4670383A (en) * 1984-09-13 1987-06-02 Boehringer Mannheim Gmbh Immune-chemical measurement process for haptens and proteins

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4427782A (en) * 1981-03-03 1984-01-24 Caldwell Harlan D Isolation of principal outer membrane protein and antigen of Chlamydia trachomatis
US4544640A (en) * 1982-04-09 1985-10-01 Fujizoki Pharmaceutical Co., Ltd. Anti immune complex antibody for determining SLE, rheumatoid arthritis or tetanus
US4636478A (en) * 1984-07-16 1987-01-13 Becton, Dickinson And Company Monoclonal antibodies recognizing L-thyroxine
US4670383A (en) * 1984-09-13 1987-06-02 Boehringer Mannheim Gmbh Immune-chemical measurement process for haptens and proteins

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
H. EISEN, "Immunology", Second Edition, published 1980, by Harper & Row, Publishers (Philadelphia, Pennsylvania, USA), see page 343, column 2 and figure 17-8. *
Proceedings of the National Academy of Science, USA, Vol. 79, issued June 1982 (Washington, D.C., USA), D.A. NEMAZEE et al., "Enhancing Antibody: A Novel Component of the Immune Response", see page 3832, column 1. *
The Journal of Biological Chemistry, Vol. 252, No. 3, issued 10 February 1977, (Bethesda, Maryland, USA), D.W. CLEVELAND et al., "Peptide Mapping by Limited Proteolysis in Sodium Dodecyl Sulfate and Analysis by Gel Electrophoresis", see page 1105, column 1. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0264219A2 (fr) * 1986-10-09 1988-04-20 Syntex (U.S.A.) Inc. Récepteurs pour des complexes immunologiques, leurs procédés de préparation et méthodes d'essais les utilisant
EP0264219A3 (en) * 1986-10-09 1990-04-11 Syntex (U.S.A.) Inc. Receptors for immune complexes, their methods of production, compositions containing them, assay methods and assay kits using them
US6326159B1 (en) 1986-10-09 2001-12-04 Dade Behring Marburg Gmbh Receptors for immune complexes
US20110086364A1 (en) * 2008-06-12 2011-04-14 Teknologian Tutkimuskeskus Vtt Detection of cannabis use
US8518653B2 (en) * 2008-06-12 2013-08-27 Teknologian Tutkimuskeskus Vtt Detection of cannabis use
EP2168985A1 (fr) 2008-09-30 2010-03-31 Siemens Healthcare Diagnostics Products GmbH Anticorps destinés à la détermination du fragment de prothrombine F2/F1+2 dans un test d'immunité homogène
DE102008049601A1 (de) 2008-09-30 2010-04-01 Siemens Healthcare Diagnostics Products Gmbh Antikörper zur Bestimmung des Prothrombin-Fragments F2/F1+2 in einem homogenen Immunoassay
US9096674B2 (en) 2008-09-30 2015-08-04 Siemens Healthcare Diagnostics Products Gmbh Antibodies for determining the prothrombin fragment F2/F1+2 in a homogeneous immunoassay

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